1. Field of the Invention
This invention relates to nucleic acid and amino acid sequences which regulate the polyketide toxin cercosporin, gene constructs, and methods related thereto. It further relates to the use of a nucleic acid to genetically engineer plants for resistance to the toxin.
2. Description of the Prior Art
Fungi of the genus Cercospora are widespread, economically important pathogens of a diverse array of crop plants, including for example, banana, sugar beet, coffee, tobacco, corn, sorghum, peanut, and soybean (Agrios, Plant Pathology, 3rd Ed., Academic Press, San Diego, Calif., 356-357, 1988). Resistance to the phytotoxin, cercosporin, produced by many species of plant pathogenic Cercospora fungi, has not been found in any commercial crops. Cercosporin is the major disease factor in purple-seed stain of soybeans and other crop diseases caused by this fungus. United States corn growers have experienced increasingly serious outbreaks of gray leaf spot disease due to Cercospora zeae-maydis. The lack of highly resistant plant cultivars, particularly early maturing corn in the Midwest, is crucial. With a total United States value of over $16 billion, corn growers, state economies, and commercial hybrid seed producers face potentially dramatic economic loss due to gray leaf spot. The situation with soybeans, while significant, is less dramatic. Soybean yield losses due to purple-seed stain have averaged about 1-2% or less in recent years. But the United States soybean crop is now valued at over $11 billion. The presence of more than 5% purple-stained beans at market results in significantly lower grower prices due to lower bean quality and the additional processing required for purple-stained beans. Furthermore, substantial crop losses have been attributed to the leaf phase of the disease in the southern United States when weather conditions favor the disease. One new approach to crop disease management is the use of pathogen-derived genes for resistance.
Scientists have traditionally used cross-breeding and hybridization techniques to provide plants having particular desired traits such as increased hardiness, nutritional value, taste, appearance, and disease resistance, etc., but these techniques are at best lengthy, time- consuming processes which do not necessarily result in achievement of a particular goal. With soybean, an oilseed of major importance to the world's economy, the search for enhanced or durable resistance to Cercospora is complicated by the fact that cultivar susceptibility to foliar and seed infections have no strong relationship. Indeed, some cultivars show resistance to seed stain and susceptibility to leaf blight. The advent of genetic engineering provides the opportunity to introduce genetic material directly into a plant, which upon expression in the plant, would result in a plant with resistant to cercosporin.
Polyketides such as cercosporin, for example, are products of secondary metabolism in bacteria, fungi, and plants. This group of compounds includes important bacterial and fungal antibiotics, plant flavonoids and fungal mycotoxins and phytotoxins (Hopwood et al, Annual Review of Genetics, Volume 24, 37-66, 1990). Many phytopathogenic fungi of the genus Cercospora produce the red polyketide toxin, cercosporin (Daub, Phytopathology, Volume 72, 370-374, 1982; Lynch et al, Trans. Br. Mycol. Soc., Volume 69, 496-498, 1977) which was first isolated from C. kikuchii (Kuyama et al., J. Am. Chem. Soc., Volume 79, 5725-5762, 1957). The structure of cercosporin, a red perylene quinone derivative, [1,12-bis(2-hydroxy-propyl)-2,11 dimethoxy-6,7-methylenedioxy-4,9-dihydroxyperylene-3,10-quinone; Molecular Weight: 534] was determined independently by Lousberg et al (J. Chem. Soc. Chem. Commun., 1971:1463-1464, 1971) and Yamazaki et al (Agric. Biol. Chem., Volume 36, 1707-1718, 1972). Cercosporin is a non-host-specific toxin, which, in the presence of light, interacts with molecular oxygen to produce both superoxide radicals and singlet oxygen (Daub et al, Plant Physiol., Volume 73, 855-857, 1983). These activated oxygen species cause peroxidation of cell membrane lipids resulting in electrolytic leakage, a decrease in membrane fluidity and cell death (Daub, ACS Symp. Ser., Volume 339, 271-280, 1987). Several lines of evidence indicate that cercosporin plays an essential role in Cercospora pathogenicity: high light intensity is absolutely required for both disease development (Calpouzos, Ann. Rev. Phytopathology, Volume 4, 369-390, 1966; Calpouzos et al, Phytopathology, Volume 57, 799-800, 1967) and toxin action (Daub, 1982 supra), toxin can be isolated from naturally infected tissues (Fajola, Physiol. Plant Pathol., Volume 13, 157-164, 1978; Upchurch et al, Appl. Environ. Microbiol., Volume 57(10), 2940-2945, 1991); application of the toxin alone can produce disease symptoms on host plants (Balis et al. Phytopathology, Volume 61, 1477-1484, 1971; Fajola, 1978, supra), and non-toxin-producing mutants of Cercospora kikuchii fail to induce disease symptoms in soybean plants (Upchurch, 1991, supra). Although little is known about the biosynthesis of cercosporin, results from nuclear magnetic resonance and mass spectrometry analysis have indicated a polyketide route of synthesis and one unstable polyketomethylene intermediate has been proposed but not isolated (Okubo et al, Agric. Biol. Chem., Volume 39, 1173-1175, 1975). No enzymes or chemical intermediates in the cercosporin biosynthetic pathway have been identified. The identification and isolation of a gene responsible for conferring a major level of resistance to cercosporin-producing microorganisms would allow the development of crops resistant to fungal diseases caused by this toxin. Although there are no reports of resistance to cercosporin in crop plants, Batchvarova et al (Phytopathol., Volume 82, 1477-1484, 1992) disclose a cercosporin resistance in a common weed. The annual weed, Louisiana red rice is resistant to all known races of Cercospora oryzae and has a resistant to cercosporin. In sensitive rice plants, cercosporin was demonstrated to accumulate in plant tissue, a phenomenon which has been seen in soybean. It was hypothesized that the resistance seen in Louisiana red rice is due to a combined effect of active efflux of the toxin from resistant cells possibly associated with cercosporin degradation or the action of carotenoids in quenching active oxygen species. U.S. Pat. No. 5,262,306 (Robeson et al) discloses cercosporin-resistant bacteria that have the ability to degrade cercosporin. The patent also states that the gene responsible for this cercosporin-degrading characteristic could be isolated and cloned in an appropriate vector and inserted into a plant.
Cercosporin-resistant crop plants have not been discovered to date. Therefore, the development of transgenic cercosporin-resistant plant varieties would be a useful approach to the control of Cercospora-induced plant diseases. The present invention, described below, provides a direct means to genetically engineer plants with resistance to this universally toxic polyketide, cercosporin, which is different from the prior art.